The Section on Molecular Signal Transduction investigates signal transduction pathways that mediate the actions of hormones, growth factors and neurotransmitters in mammalian cells, with special emphasis on the role of phosphoinositide-derived messengers. Phosphoinositides are a small fraction of the cellular phospholipids, but have critical roles in the regulation of many (if not all) signaling protein complexes that assemble on the surface of cellular membranes. Phosphoinositides regulate protein kinases and GTP-binding proteins, as well as membrane transporters including ion channels, thereby controlling many cellular processes such as proliferation, apoptosis, metabolism cell migration and differentiation. This group focuses on one family of enzymes, the phosphatidylinositol 4-kinases (PI4Ks) that catalyze the first committed step in phosphoinositide synthesis. Current studies are aimed at (1) understanding the function and regulation of several phosphatidylinositol (PI) 4-kinases in the control of the synthesis of hormone-sensitive phosphoinositide pools; (2) characterizing the structural features that determine the catalytic specificity and inhibitor sensitivity of PI 3- and PI 4-kinases; (3) defining the molecular basis of protein-phosphoinositide interactions via the pleckstrin homology and other domains of selected regulatory proteins; (4) developing tools to analyze inositol lipid dynamics in live cells; (5) determining the importance of the lipid-protein interactions in the activation of cellular responses by G protein-coupled receptors and receptor tyrosine kinases.[unreadable] [unreadable] Sphingomyelin (SM) is a critical lipid component of the plasma membrane that together with cholesterol and glycolipids forms a special liquid-ordered microdomain of cellular membranes often referred to as rafts. Rafts concentrate many signaling proteins and also contain inositol phospholipids and hence are considered to be active zones in signal transduction. The regulation of cholesterol and sphingomyelin metabolism is intimately interrelated but relatively little is known about the regulatory pathways that link them together. Efficient synthesis of sphingomyelin in the Golgi has been shown to require the steady supply of ceramide from the ER by a process distinct from the vesicular transport between the two organelles. The mechanism of this transport has been recently revealed by the identification of a lipid transport protein, named CERT. CERT has a lipid binding START domain at the C-terminus, a PH-domain at the N-terminus and a so-called FFAT (diphenylalanine in an acidic track) domain that binds the ER-localized protein, VAP-A. The START domain is both necessary and sufficient for ceramide binding and transport, yet a mutation within the PH domain renders the molecule unable to fulfill its transport function pointing to the PH domain as a critical component for the docking/regulation of the molecule. The CERT PH domain shows a high degree of similarity to PH domains that specifically recognize phosphatidylinositol 4-phosphate (PtdIns4P), such as those of the OSBP, FAPP1 and FAPP2 proteins, and has been shown to localize to the Golgi. This finding indicates that PtdIns4P and PI4K enzymes are likely to regulate the transport function of CERT.[unreadable] Given the presence of multiple PI4Ks in the Golgi and one in the ER, it was of interest to determine which of these enzymes (if any) are important in supporting the transport of ceramide between the ER and the Golgi. For this a combination of pharmacological and genetic approaches was used and the transport of exogenously added fluorescent ceramide analogues to the Golgi as well as the fate of the endogenous ceramide labeled with [3H]-serine were followed. These studies identified PtdIns4P as a critical regulatory lipid in the Golgi facilitating ceramide transport and PI4KIIIbeta the enzyme that controls this process. The significance of this finding is that it points out the importance of inositide lipid-mediated regulation of the transport and metabolism of major phospholipids in mammalian cells a hitherto unrecognized function of phosphoinositide kinases. Future research will address the question of whether cholesterol metabolism is also affected by PI4K enzymes.[unreadable] [unreadable] Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is the major phosphoinositide species in mammalian cells that has been associated with numerous molecular events critical for cellular signaling. PtdIns(4,5)P2 is hydrolyzed by phospholipase C enzymes to generate DAG and Ins(1,4,5)P3, two pivotal second messenger. PtdIns(4,5)P2 is also converted by Class I PI 3-kinases to PtdIns(3,4,5)P3, another important membrane-bound messenger molecule. PtdIns(4,5)P2 directly interacts with several ion channels, transporters, actin binding proteins, and regulates enzymes such as PLC and PLD. A number of molecules that are part of the receptor internalization machinery have also been shown to contain inositide binding domains but the exact lipid species that regulates them in the cell has not been firmly established. It is a major challenge to understand how a single type of molecule is able to regulate so many processes simultaneously and perhaps independently within the plasma membrane (PM). [unreadable] We developed a strategy to promptly regulate membrane PtdIns(4,5)P2 levels by a drug-inducible membrane targeting strategy based on the heterodimerization of the FRB domain of mTOR and FKBP12. In this approach the enzyme of interest ? in this case a type-IV phosphoinositide 5-phosphatase (5-ptase) is fused to the FKBP12 protein and upon addition of rapamycin (Rapa) (or an analogue that does not interact with endogenous proteins) the enzyme rapidly translocates to the membrane where its binding partner, the FRB?domain is targeted. [unreadable] Rapa-induced PM recruitment of a truncated type-IV 5-phosphatase containing only the 5-phosphatase domain fused to FKBP12, rapidly decreased PM PtdIns(4,5)P2 as monitored by the PLC?1PH-GFP fusion construct. This decrease was paralleled by rapid termination of the ATP-induced Ca2+ signal and the prompt inactivation of menthol-activated TRPM8 channels. Depletion of PM PtdIns(4,5)P2 was associated with a complete blockade of transferrin uptake and inhibition of EGF internalization. None of these changes were observed upon Rapa-induced translocation of an mRFP-FKBP12 fusion protein that was used as a control.